The Melanocortin Receptor 4 (MC4R) is a G-protein coupled receptor (GPCR) which is central to the control of appetite. Upstream signaling pathways converge to control release of the agonist of MC4R, alpha-melanocyte stimulating hormone (MSH), which transduces the signal of satiety upon binding to the receptor. Like other GPCRs, MC4R is desensitized upon agonist stimulation, leading to decreased response to a subsequent signal of equal magnitude. Our lab has shown that MC4R is not desensitized according to the canonical desensitization mechanism of most GPCRs. Classically, GPCRs are phosphorylated and rapidly endocytosed upon agonist stimulation. As we have shown, MC4R is internalized constitutively, at the same rate in the presence and absence of the agonist. Upon agonist stimulation, less receptor is recycled back to the plasma membrane, likely accounting for decreased expression of the receptor at the cell surface. I have shown that the decrease in surface receptor upon incubation with MSH leads to a corresponding decrease in cAMP production in the wild type MC4R. Conversely, a mutated MC4R with presumed phosphorylation sites mutated to alanine has similar cell surface expression, recycles back to the plasma membrane at the same rate, and, as I have shown, produces equivalent levels of cAMP regardless of treatment with the agonist. This result suggests that receptor phosphorylation is necessary to sequester the receptor in intracellular compartments in the presence of the agonist. Because desensitization of MC4R would lead to increased appetite, inhibiting factors which function in this pathway is a potential target for obesity drugs. In this study, we will idenify factors involved in sorting phosphorylated MC4R away from recycling pathways and the kinase(s) that phosphorylate MC4R. In Aim1 we will perform an affinity purification of a stably transfected HA-MC4R-GFP and determine interacting factors via mass spectrometry. Co-purified proteins will be investigated for their possible effect on MC4R traffic and desensitizatio. In Aim 2, we will silence or pharmacologically inhibit G- Protein Coupled Receptor Kinases (GRKs) and kinases that have been previously described to be activated upon MC4R activation, and measure the effect on the phosphorylation state, plasma membrane expression and activity of MC4R. By identifying and factors involved in sequestering it in intracellular compartments, and factors that phosphorylate MC4R we will create drug targets for much needed obesity therapy. PUBLIC HEALTH RELEVANCE: Obesity is an increasingly important health problem in the United States and worldwide. Although effective treatments for the prevention and reversal of obesity would have an enormous public health benefit, currently available therapies are largely ineffective or unsafe. Here, we propose to study the desensitization of the Melanocortin Receptor 4, a protein central to the control of appetite and energy expenditure, in an attempt to identify novel drug targets for the prevention and treatment of obesity.